Project Abstract Innate immunity begins with detection of infection by pattern recognition receptors (PRRs). PRRs are located in all cellular compartments, allowing the identification of all potential pathogenic invasions. The sensing mechanisms of extracellular pathogens by Toll-like Receptors is well-defined, and their localization clearly optimizes their ability to detect these pathogens. However, many PRRs reside within the host cell cytosol, and the mechanisms that control their ability to efficiently sense infection and their localization within cells remain unknown. One of these cytosolic PRRs, RIG-I, is a key sensor of RNA virus infection, binding viral RNA (vRNA) directly. Activation of RIG-I occurs rapidly upon infection, sensing many viruses before their replication cycle initiates. This suggests that RIG-I binds vRNA from incoming viral particles, despite its extremely low expression prior to activation. RIG-I is thought to interact with its ligand by diffusion, as both are present in the cytosol. However, this does not account for how rapidly RIG-I senses its ligand and points out a lack of understanding of the initial steps in sensing infection. Given these rapid mechanics, RIG-I may be positioned to sense viral infection at its onset. As entry is the earliest point of vRNA exposure to the cytosol, RIG-I is poised likely at sites of viral entry. The goal of this project is to discover the initial site of vRNA binding by RIG-I, identify the regulation of this phenomenon, and determine its impact on the efficiency of RIG-I signaling. This project will fully characterize the subcellular location of RIG-I and determine which portions of RIG-I mediate this localization. Furthermore, the triggers for RIG-I localization through ligand interaction or post translational modifications will be identified. Finally, the role of RIG-I localization in its ability to detect viral infection will be directly tested. Understanding where this recognition event occurs will provide key insight into the kinetics of host-pathogen interaction. In addition, this project will characterize the location of vRNA recognition by RIG-I, elucidating the spatiotemporal regulation of this extensively studied pathway.